Controlling the stereochemistry of chemical reactions is a vital skill in the small molecule drug discovery and development process. The identification of catalysts that promote the formation of the desired stereo centers is paramount, yet has been a limiting step in the synthesis of the complex chemical structures found in drugs today. We propose to establish a general approach that will enable the creation of de novo biocatalysts for a wide range of chemical reactions. Our strategy is different as it goes well beyond current enzyme engineering methods that are limited to the optimization of existing enzymes. We will isolate active biocatalysts from combinatorial libraries of 1013 randomized proteins through an in vitro selection and evolution technique that we have recently pioneered. The key advantage is the use of libraries that contain several orders of magnitude more protein variants than other protein engineering methods. Our specific aims are: 1) to isolate de novo biocatalysts from a library based on nature's most successful enzyme scaffold, the (/)8 barrel fold. 2) To identify features necessary to build the most efficient biocatalysts by comparing the (/)8 barrel enzyme library with a non-catalytic zinc finger scaffold library for the selection of novel activites. 3) To identify the evolutionary potential of an artificial biocatalyst by optimizing its activity ad stability through in vitro evolution. In order to demonstrate the broad applicability of our selecton approach, we will focus on two important, but vastly different bond formation reactions: a carbon-carbon bond formation between small molecules and a phosphodiester bond formation between two RNA oligonucleotides. We will isolate biocatalysts for a Diels- Alder reaction, one of the central reactions in organic chemistry that creates up to four new stereo centers and is therefore critical for the synthesis of many high-value polycyclic natural products in pharmaceutical chemistry. Because of the importance of the Diels-Alder reaction, several catalyst design efforts have targeted this chemistry in the past. Therefore, this reaction serves as a benchmark to evaluate our broadly applicable technology. Furthermore, we will generate an artificial RNA ligase enzyme that has unique potential as a medical research tool to advance RNA sequencing applications for certain classes of RNA. Preliminary studies show that our approach is feasible. In a proof of concept experiment, we created an artificial biocatalyst that catalyzes a reaction for which there are no known natural enzymes. This highly selective de novo catalyst accelerates the reaction more than two-million-fold. The long-term goal of our research is to create biocatalysts that facilitate key reactions in the drug synthesis process for which no catalyst is available. We envision our general method for producing designer catalysts for chemical reactions of interest to substantially impact the way drug synthesis is approached.